Whole genome sequencing insight into carbapenem-resistant and multidrug-resistant Acinetobacter baumannii harboring chromosome-borne blaOXA-23

ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat to hospitalized patients as effective therapeutic options are scarce. Based on the genomic characteristics of the CRAB strain AB2877 harboring chromosome-borne blaOXA-23, which was isolated from the bronchoalveolar lavage fluid (BALF) of a patient in a respiratory intensive care unit (RICU), we systematically analyzed antibiotic resistance genes (ARGs) and the genetic context associated with ARGs carried by CRAB strains harboring chromosome-borne blaOXA-23 worldwide. Besides blaOXA-23, other ARGs were detected on the chromosome of the CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme). Several key genetic contexts associated with the ARGs were identified on the chromosome of the CRAB strain AB2877, including (1) the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id (2); the resistance island AbGRI3 harboring armA and mph(E)-msr(E) (3); the Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia; and (4) the structure “ISAba1-blaADC-25.” The first two genetic contexts were most common in ST195/1816, followed by ST208/1806. The last two genetic contexts were found most frequently in ST208/1806, followed by ST195/1816. IMPORTANCE The blaOXA-23 gene can be carried by plasmid or chromosome, facilitating horizontal genetic transfer and increasing carbapenem resistance in healthcare settings. In this study, we focused on the genomic characteristics of CRAB strains harboring the chromosome-borne blaOXA-23 gene, and the important genetic contexts associated with blaOXA-23 and other ARGs were identified, and their prevalent clones worldwide were determined. Notably, although the predominant clonal CRAB lineages worldwide containing the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id was ST195/1816, followed by ST208/1806, the CRAB strain AB2877 in our study belonged to ST208/1806. Our findings contribute to the knowledge regarding the dissemination of CRAB strains and the control of nosocomial infection.

infections in patients in the ICU (3).The success of A. baumannii as a nosocomial pathogen is likely attributable to its high antibiotic resistance (4).
The emergence of multidrug-resistant (MDR) and extremely drug-resistant (XDR) A. baumannii has made it one of the most troublesome nosocomial pathogens worldwide in recent years (5).Data from the China Antimicrobial Surveillance Network (CHINET) indicate that the resistance levels of A. baumannii isolates to imipenem and meropenem will remain relatively stable (accounting for more than 75%) from 2018 to 2022 (6).Indeed, in 2017, the World Health Organization listed CRAB as a priority one critical pathogen, with an urgent need for the development of new treatments (7).
Horizontal gene transfer and mutational changes in chromosomal structures are the main causes of multidrug-resistant (MDR) and extreme drug-resistant (XDR) A. baumannii (8).The spread of antibiotic resistance genes (ARGs) is facilitated by mobile genetic elements, such as plasmids, insertion sequences (ISs), transposons, and integrons (9).To better understand the characteristics of ARGs and the genetic environment associated with ARGs carried by the A. baumannii strain AB2877, which was isolated from a patient in a tertiary hospital, we performed in silico typing and comparative analysis of the A. baumannii strain AB2877 with other A. baumannii strains available in the NCBI database.This study highlights the important relationship between ARGs and mobile genetic elements in A. baumannii that harbor chromosome-borne bla OXA-23 .

Genetic context associated with armA and mph(E)-msr(E)
armA and mph(E)-msr(E) were located on a ~11.5-kbTn6180-derived fragment of the resistance island AbGRI3, which was bracketed by ISEc28 and IS26 (Fig. 2A).ISEc28 was inserted upstream of the armA gene.For the macrolide resistance genes (mph(E)-msr(E)), an IS4-like element ISEc29 was inserted upstream of the mph(E)-msr(E), and an IS66-like element ISAba24 was inserted downstream of the mph(E)-msr(E) (Fig. 2A).

Genetic context associated with bla TEM-1D and aph(3')-Ia
bla TEM-1D and aph(3')-Ia were located on a 9.4-kb Tn3-like composite transposon, which was bracketed by two copies of IS26 in the same orientation (Fig. 3A).Additionally, four copies of IS26 in the same orientation were found in the 9.4-kb Tn3-like composite transposon.The bla TEM-1D gene was flanked by two direct repeats of IS26, and the aph(3')-Ia gene was also flanked by two direct repeats of IS26 (Fig. 3A).

DISCUSSION
In this study, we describe a CRAB strain, AB2877, belonging to ST208/1806 (Oxford MLST scheme) or ST2 (Pasteur MLST scheme), which was isolated from the bronchoalveolar lavage fluid (BALF) of a 66-year-old patient in the respiratory intensive care unit (RICU).Among the antibiotics tested, the CRAB strain AB2877 was only susceptible to colistin and cefiderocol, indicating their great clinical value in the treatment of CRAB-causing infections.The CRAB strain AB2877 carries chromosomal bla OXA-23 gene, which is a key determinant of antibiotic resistance found in Acinetobacter species, which encodes a class D β-lactamase enzyme that confers resistance to carbapenem antibiotics (10).The bla OXA-23 -like gene is prevalent in China and is present in 97% of carbapenem-resistant isolates (11).ISAba1 is a strong promoter sequence that influences the expression of neighboring genes, including those encoding antibiotic resistance determinants (12).Upstream of resistance genes, ISAba1, can significantly increase their expression, leading to increased resistance to antibiotics (13).
In CRAB AB2877, the bla OXA-23 gene was embedded in Tn2006, which is bracketed with two copies of ISAba1 in different orientations.The transposon Tn2006 is one of the most common transposons worldwide (14) and may be one of the main reasons for the global dissemination of carbapenem resistance in A. baumannii (15).In particular, besides transposon Tn2006, transposons Tn2008 and Tn2009 also appeared to contribute significantly to the dissemination of bla OXA-23 in China (16)(17)(18).Furthermore, we found that Tn2006 harboring bla OXA-23 was present not only on the chromosomes but also on the plasmids of A. baumannii, as well as other pathogens, such as K. pneumoniae and Proteus mirabilis (19), suggesting that several species may be reservoirs or scatterers for this class D carbapenemase gene.In addition to the bla OXA-23 gene, other beta-lacta mase genes (bla OXA-66 , bla ADC-25 , and bla TEM-1D ) were also detected in this study.This finding is consistent with those of previous reports.For example, an A. baumannii isolate has been reported to harbor bla OXA-23 , blaOXA-66, and bla ADC-25 in south China (20).Several coexisting multi-beta-lactam resistance genes contribute significantly to the extensive drug resistance observed in A. baumannii strains, which poses significant challenges for infection control and clinical management.
In this study, the CRAB strain AB2877 belonged to ST208/1806 (Oxford MLST scheme).Analysis of the genetic context associated with detected ARGs indicated that the genetic context associated with bla TEM-1D and that of bla ADC-25 were easily detected in ST208/1806, followed by ST195/1816.The MDR regions associated with bla OXA-23 , tet(B)-tetR(B), aph(3'')-Ib, aph (6)-Id, and the resistance island AbGRI3 harboring armA and mph(E)-msr(E) were commonly detected in ST195/1816, followed by ST208/1806.ST208 is widely recognized as a predominant lineage of A. baumannii GC2 worldwide, and some researchers believe that ST208 may have originated in North America and evolved into two clades (21).Reports of carbapenem-resistant A. baumannii ST195 in China have increased gradually in recent years (22,23).
The bla OXA-23 -like gene is almost always found within transposons and is commonly associated with an antibiotic-resistance genomic island (AbGRI) (14).The ribosomal RNA methyltransferase gene armA, an aminoglycoside resistance gene located on the resistance island AbGRI3, has been widely reported in A. baumannii (24).In the AB2877 isolate, the genetic contexts associated with armA and mph(E)-msr(E) were similar to those of Tn6180.Tn6180-borne armA or AbGRI3 has spread worldwide, especially in Japan and East Asia (25,26).
In this study, we describe the genomic characteristics of the multidrug-resistant CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme) harboring chromosomeborne bla OXA-23 , which was isolated from the BALF of a patient in the RICU in China.Several key genetic contexts associated with bla OXA-23 and other ARGs were found on the chromosome of the CRAB strain AB2877.Based on the genomes of A. baumannii available in the GenBank database, we explored the predominant clonal lineages of A. baumannii worldwide, including different genetic contexts of the CRAB strain AB2877.The bla OXA-23 gene was located in the MDR region associated with bla OXA-23 , tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id, which was most commonly found in ST195/1816, followed by ST208/1806.Furthermore, the resistance island AbGRI3 that harbors armA and mph(E)-msr(E) carried by strain AB2877 was most frequently found in CRAB ST195/1816, followed by CRAB ST208/1806.The CRAB strain AB2877 also carried one Tn3-like composite transposon bracketed by two copies of IS26 containing bla TEM-1D and aph(3')-Ia and one structure "ISAba1-bla ADC-25 , " which were most commonly found in ST208/1806, followed by ST195/1816.

Funder Grant(s) Author(s)
Zhuhai City Science and Technology Plan Project ZH22036201210092PWC Wei Wang Xiangshan Talent Project of Zhuhai People's Hospital 2020XSYC-02 Xiaobin Li

FIG 3 ( 7 FIG 4
FIG 3 (A) Genetic structures associated with bla TEM-1D and aph(3')-Ia.Genome sequences used to draw the diagrams from GenBank (A. baumannii F11, A. baumaannii AB329 chromosome, A. baumannii AB2877 chromosome, A. baumannii X4-300 plasmid, and Acinetobacter sp.FDAARGOS_560 chromosome).Genes and ORFs are shown as arrows, and the direction of transcription is indicated by arrowheads.The resistance and transposase genes are shown in red and blue, respectively.(B) The unrooted tree shows the Top 5 most prevalent MLST from 156 strains of A. baumannii.The phylogenetic tree was created using the kSNP v3.1 based on the whole genomes of 156 strains of A. baumannii, which harbored the genetic structures associated with bla TEM-1D and aph(3')-Ia.The five most common MLST genotypes are ST208/1806 (red), ST195/1816 (green), ST350! (blue), ST451/1809 (purple), and ST381! (orange).